Chemiluminescent acridinium compounds have emerged to be extremely useful labels for immunoassays and nucleic acid assays. Hydrophilic acridinium esters containing N-sulfopropyl groups (NSP) are useful for protein labeling as well as for the preparation of small molecule conjugates, as described by Law et al. in U.S. Pat. No. 5,656,426 and Natrajan et al. in U.S. Pat. No. 6,664,043, the disclosures of which are hereby incorporated by reference. Furthermore, acridinium ester labels containing NSP groups display improved aqueous solubility compared to the corresponding N-methyl analogs. These hydrophilic acridinium esters offer improved performance in immunoassays. For example, chemiluminescent acridininum dimethylphenyl esters containing NSP groups have been used as labels in automated immunoassays in Siemens Healthcare Diagnostics' ADVIA: Centaur® systems. These labels exhibit excellent chemiluminescent stability compared to unsubstituted acridinium phenyl esters (Law et al, J. Biolumin. Chemilumin., 1989, 4, 88-98). Improvements to the specific binding of these chemiluminescent labels to an analyte have also been made by incorporating poly(ethylene)glycol (PEG) or zwitterions in the acridinium ester structure (Natrajan et al, Org. Biomol. Chem., 2011, 9, 5092-5103; Natrajan et al, Anal. Biochem., 2010, 406, 204-213).
The synthesis of acridinium esters containing N-sulfopropyl groups entails N-alkylation of the corresponding acridine esters with the potent carcinogen 1,3-propane sultone. In general, this reaction is carried out by heating the acridine ester with a vast excess of propane sultone in the absence of solvent, as described by Law et al in U.S. Pat. No. 5,656,426, incorporated by reference herein. These harsh conditions for the N-alkylation reaction are necessitated by the poor reactivity of the hindered acridine nitrogen towards alkylating reagents.
A major disadvantage of this reaction is that 1,3-propane sultone is quite toxic and poses a significant health hazard (Bolt and Golka, Toxicol. Lett., 2004, 151, 251-254; Ulland et al, Nature, 1971, 230, 460-461). As reported recently by Bolt and Golka in Toxicology Lett. 2004, 151, 251-254, the disclosure of which is incorporated by reference, propane sultone is directly alkylating, genotoxic and carcinogenic. Bolt and Golka observe that “malignancies observed within a group of persons exposed to 1,3-propane sultone appear surprisingly consistent with the expectations from the available animal studies” and are characterized by latency times up to 30-40 years or more after limited periods of past exposure. Thus, chemical transformations under reaction conditions that limit the use this toxic reagent are desirable.
More recently, Natrajan and Wen, in Green Chem., 2011, 13, 913-921 and U.S. Patent Application Publication No. 2010/0256380 described a synthetic protocol for the N-alkylation of acridine esters in ionic liquids. The increased reactivity of the acridine ester precursors with 1,3-propane sultone in ionic liquids provide for the synthesis of a variety of functionalized acridinium esters containing N-sulfopropyl groups with reduced side reactions or decomposition. Although this synthetic protocol in ionic liquids reduced the amount of 1,3-propane sultone necessary for N-alkylation of the acridine compound, sultones, particularly the highly toxic 1,3-propane sultone, remained a necessary part of the reaction.
The above cited literature points to the general difficulty in introducing the N-sulfopropyl group in acridine compounds which necessitates conducting the reaction using highly reactive alkylating reagents. It is important to eliminate the use of the carcinogenic reagent 1,3-propane sultone altogether in the synthesis of acridinium esters with N-sulfopropyl groups. It is therefore an object of the invention to provide a synthetic process for N-alkylating an acridine compound using a suitable and relatively benign alkylating agent to achieve this chemical transformation in good yields.